DNA damage due to voluntary or involuntary exposure to various environmental genotoxic agents (e.g. irradiation and chemical carcinogens) is a major cause of human cancers and many other human diseases. In response to DNA damage, cells activate several major DNA damage response (DDR) pathways such as DNA damage checkpoints, DNA repair, and apoptosis. ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) are the two major DNA damage checkpoint protein kinases which play critical roles in DDR by signaling DNA damage, activating checkpoints, arresting cell cycle progression and facilitating DNA repair to restore DNA integrity. Both ATM and ATR are protein kinases belonging to the phosphoinositide 3-kinase-related protein kinases (PIKK) family and are sensors of cellular stress. Intriguingly, however, a body of evidence from mouse model and human epidemiologic studies show that unlike ATM whose deficiency promotes carcinogenesis, ATR inhibition suppresses carcinogenesis. This is puzzling given the general cancer-suppression role of DNA damage checkpoints. In this project, we will test the hypothesis that (1) besides functioning as a tumor suppressor via its checkpoint regulatory role in the nucleus against moderate DNA damage, ATR in the cytoplasm can function as an anti-apoptotic protein at mitochondria upon severe DNA damage; (2) this anti-apoptotic activity in the cytoplasm occurs via direct involvement of ATR in regulating the DNA damage-induced mitochondria cell death pathways in a checkpoint-independent manner; and (3) this transformation of ATR from tumor suppressor to anti-apoptotic protein is regulated by a prolyl isomeric modification of the cytoplasmic ATR. The modification changes the conformation of ATR, making it anti-apoptotic so that its inhibition suppresses carcinogenesis. These hypotheses will be tested in the following specific aims. Aim 1: To define the UV-induced modification of cytoplasmic ATR and its cellular effects; Aim 2: To determine the mechanisms by which the functions of cytoplasmic ATR are regulated; and Aim 3: To determine the molecular basis of the ATR anti-apoptotic activity at mitochondria. The proposed studies represent an innovative effort highly relevant to cancer and other human diseases such as neurodegenerative and cardiovascular diseases.